Reverse transcriptase (RT) is an RNA-dependent DNA polymerase that synthesizes DNA using RNA as a template. It has been an indispensible reagent in molecular biology for the study of RNA, and in molecular diagnostics for determining the identity of an organism based on a specific RNA sequence in conjunction with DNA amplification. Commonly used RTs are from avian myeloblastosis virus (AMV) and Maloney murine leukemia virus (M-MuLV) and their derivatives. Although each of these RTs has advantages in certain applications, they also have limitations. For example, in molecular diagnostics, the primary concerns are sensitivity and reaction speed. Sensitivity requires that the RT be able to generate enough cDNA for a given amplification platform; the reaction speed determines how quickly the required cDNA product is produced.
Loop-mediated isothermal amplification (LAMP) has been recently adapted to molecular diagnostics for many pathogens due to its convenience in detection and high sensitivity. When a RT is included in LAMP (reverse transcription-LAMP, RT-LAMP), it can be efficiently applied to detect RNA targets, and it has been successfully used for the detection of a number of RNA viruses with great sensitivity. In RT-LAMP, it is essential that the RT be able to efficiently synthesize DNA using the target RNA under conditions optimized for DNA amplification by a DNA-dependent DNA polymerase. This is a significant hurdle which substantially impacts RT selection in RT-LAMP, because the optimal reaction conditions for most RTs do not match the optimal reaction conditions for DNA amplification by DNA-dependent DNA polymerases. The RT most typically used in RT-LAMP is from AMV, because it affords reasonable sensitivity and reaction speed.
Polymerase chain reaction (PCR) has been a major player in DNA amplification. Similar to RT-LAMP, by inclusion of a RT in a PCR reaction (RT-PCR), it is possible to detect RNA. Traditionally RT-PCR is performed in two steps: the first step is RT in an optimized buffer and then PCR in a second step in another buffer condition optimized for PCR. Although these two steps can theoretically be combined, finding a single set of reaction conditions suitable for both steps is challenging. A critical issue is to find RTs that are sensitive and fast even under conditions optimized for the amplification step.
In addition, there are several other properties that would be desirable in a RT for use in one-step detection of RNA by either RT-LAMP or RT-PCR or other amplification technologies. These include: sensitivity and reaction time of the RT, tolerance to high salt and to other potential inhibitors that might carry over from previous RNA sample preparation; and enhanced thermal stability. For example, enhanced thermal stability of the RT permits reverse transcription at a higher reaction temperature so as to reduce the secondary structure of RNA and thereby increase the detection sensitivity and speed. Due to the high demand for RT in molecular diagnostics, the convenience of production and storage are also important features. For example, AMV RT is commonly produced in chicken embryos and it is well known that its production has certain limitations.